The present invention is generally in the area of bicyclic nitrone spin trapping compositions which are capable of reacting with free radicals to produce a detectable radical spin adduct.
The superoxide radical anion and the hydroxyl radical both play significant roles in the events of free radical injury to biological systems under oxidative stress. Research in recent years indicates that in most cases of oxidative damage, superoxide is the species that initiates free radical reactions that follow. Superoxide often is the precursor to the generation of the hydroxyl radical, which is considered to be most reactive oxygen-centered radical known. Therefore, detection and quantitation of superoxide anion and hydroxyl radicals in biological systems is very important in order to verify that the observed events are indeed caused by free radicals as well as to elucidate the relationship between the amount of free radical generated and its outcome. Halliwell and Gutteridge, Free Radicals in Biology and Medicine, Second Edition, Oxford, Clarendon Press, 1989; Packer and Glazer, Methods in Enzymology, Vol. 186, Oxygen Radicals in Biological Systems, Part B, Oxygen Radicals and Antioxidants, San Diego, Academic Press, 1990. Because oxygen radicals are labile in nature there is no perfect method available for their detection and quantitation.
Nitrones have been developed as useful spin traps for the detection of free radicals. The spin trapping chemistry of nitrones has been extensively reviewed, for example in: Janzen, E. G., Accounts of Chemical Research, 4, 31-40 (1971); Janzen, E. G. and Haire, D. L., Advances in Free Radical Chemistry, edited by D. D. Tanner, JAI Press Inc., Greenwich, Conn., USA Ch. 8, pp. 253-295 (1990); Perkins, M. J., Chemical Society Special Publication #24, Ch. 5, (1970); and Perkins, M. J., "Spin Trapping", in Advances in Physical Organic Chemistry, Edited by V. Gold and D. Bethell, Academic Press, New York, N.Y. Vol. 17, pp. 1-58, (1980). In general, the known reactions indicate that free radicals add to the carbon atom to produce nitroxides, and not to the oxygen atom or the nitrogen atom of the nitrone function. The nitroxides so produced have variable stability and this intrinsic stability depends on the polarity of the solvent. Kotake, Y. and Janzen, E. G., J. Am. Chem. Soc., 113, 9503-9506 (1991); Janzen, E. G. et al., Free Radical Biology and Medicine, 12, 169-173 (1992); and Janzen, E. G. et al., Tet. Lett., 33, (10) 1257-1260 (1992). Nitroxides also can be reduced or oxidized in the presence of extrinsic factors such as disproportionation reactions, redox reagents which either oxidize or reduce the nitroxide spin adduct, or subsequent additional spin trapping reactions. Janzen, E. G. et al., J. Am. Chem. Soc., 112, 8279-8284 (1990).
DMPO (5,5-dimethylpyrroline-N-oxide) has become the most commonly used spin trap in biological systems. The use of 5,5-dimethylpyrroline-N-oxide (DMPO) as a versatile spin trap was first published in: E. G. Janzen and J. I.-P. Liu, J. Mag. Res., 9:510-512 (1973). DMPO is useful to detect radicals because the hydroxyl radical spin adduct and the superoxide/hydroperoxyl radical spin adduct have distinctive EPR spectra. Janzen and Liu, Journal Magnetic Resonance, 9:510 (1973); Janzen et al., Canadian Journal Chemistry, 56:2237 (1978); Finkelstein et al., Journal American Chemistry Society, 102:4994 (1980); and Makino et al., Journal American Chemistry Society, 104:3537 (1982). These unique signatures are readily recognized in the presence of each other and thus are convenient for the study of systems where both the hydroxyl and superoxide/hydroperoxyl radicals are produced simultaneously. Moreover, since EPR spectroscopy is very sensitive, the spin trapping method permits the detection of low concentrations of these two species in in situ experiments.
The life-times for some DMPO spin adducts sometimes are low, depending on the structure of the radical. For example, the half-life of the superoxide adduct in neutral media is only about one minute. Finkelstein et al., Mol. Pharmacol., 16:676 (1979); Finkelstein et al., Arch. Biochem. and Biophys., 200:1 (1980); Janzen and Haire, in Advances in Free Radical Chemistry, D. D. Tanner, Ed., JAI Press, Greenwich, Conn., vol 1, pp 253-295 (1990); Buettner and Oberly, Biochem. Biophys. Res. Com., 83:69 (1978); Yamazaki et al., J. Biol. Chem., 265:652 (1990); Buettner, Free Rad. Res. Commun., 19, (S1), S228-S230 (1993); and Mitsuta et al., Bull. Chem. Soc. Jpn., 67:529 (1994). Therefore the intensity of the EPR signal is not proportional to the superoxide generated in the system. Also, in biological systems DMPO seems to almost exclusively concentrate in polar regions since this nitrone is very water soluble and the partition coefficient for lipid phases is very small (0.1:1). Turner and Rosen, J. Med. Chem. 29:2439-2444 (1986).
Derivatives of DMPO also have been prepared and their spin trapping chemistry explored, however the derivatives have serious drawbacks ranging from complex EPR spectra of spin adducts, to expensive necessary pathways for synthesis. Janzen et al., Can. J. Chem., 59:756-758 (1981); Rosen and Turner, J. Med. Chem., 31:428-432 (1988); Haire and Janzen, Can. J. Chem., 60:1514-1522 (1982); Janzen et al., J. Mag. Res., 9:513-516 (1973); Janzen and Zhang, J. Mag. Res., 101B:91-93 (1993); Janzen et al., J. Org. Chem., 60:5434-5440 (1995); Barasch et al., J. Am. Chem. Soc., 116:7319-7324 (1994); Janzen et al., J. Am. Chem. Soc., 116: 3738-3743 (1994) and Janzen et al., Chemistry Letters, 497-500 (1993); Frejaville et al., J. Chem. Soc., Chem. Commun., 1793-1794 (1994); and Tuccio et al., J. Chem. Soc., Perkin Trans., 2:295 (1995).
The biological applications of nitrone spin traps have been reviewed, for example in: Janzen, E. G., Free Radicals in Biology, 4, 115 (1980); and Janzen, E. G., "Spin Trapping", in "Oxygen Radicals in Biological Systems", Methods in Enzymology, L. Packer, editor, Academic Press, Inc., New York, N.Y., 105, 188-198 (1984); DeGray, J. A. and Mason, R. P. "Biological Spin Trapping" in Electron Spin Resonance, Vol. 14, "The Royal Society of Chemistry, A Specialist Periodical Report, A Review of Recent Literature to 1993" Ch. 8. pp. 246-319; Tomasi, A. and Iannone, A. "ESR Spin-Trapping Artifacts in Biological Model Systems" in Biological Magnetic Resonance, Vol. 13, EMR of Paramagnetic Molecules, edited by L. J. Berliner and J. Reuben, Plenum Press, New York, N.Y. ch. 9. pp. 353-384 (1993); and Sentjurc, M. et al., "Metabolism, Toxicity and Distribution of Spin Traps", in Nitroxide Spin Labels; Reactions in Biology and Chemistry,CRC Press, New York, N.Y. Ch. 10. pp. 199-209 (1995). These reviews mainly describe examples of the use of nitrones to detect free radicals in vitro or in vivo.
In general, nitrones with aliphatic or aryl groups attached to the nitrone function are the most useful for spin trapping. Janzen, E. G. and Haire, D. L. Advances in Free Radical Chemistry, edited by D. D. Tanner, JAI Press Inc., Greenwich, Conn., USA Ch. 8, pp. 253-295 (1990). Alicyclic nitrones have been described with the nitronyl function incorporated into the cyclic structure. Janzen, E. G. and Liu, J. I-P. J. Mag. Res., 9, 510-512 (1973); Janzen, E. G. et al., Can. J. Chem., 59, 756-758 (1981); and Zhang, Y.-K. and Janzen, E. G., Z. Naturforsch. (B), 50, (10), 1531-1536 (1995). The toxicity of nitrone compounds also has been examined. Janzen, E. G. et al., J. Biochem. Biophys. Methods, 30, 239-247 (1995).
The synthesis of tricyclic 4-azahomoadamant-4-ene N-oxides and their 1,3-dipolar cycloaddition reactions with alkynes have been reported. Yu et al., Tetrahedron Letters, 32:4965-4968 (1991). The synthesis of nitroxide derivatives of bicyclo3,2,1!-azooctane also has been described. Reznikov et al., International Conference on Nitroxide Radicals, Abstract, Sep. 18-23, 1989. The use of bicyclic nitrones as spin trapping compositions has not been explored.
There is a need for the development of spin trapping compounds for detecting free radicals in biological systems. There is a need for the development of methods for synthesizing spin trapping compounds capable of forming spin adducts with longer half-lives for use in biological systems. There also is a need for the development of methods for the synthesis of spin trapping compounds with low toxicity for animals when used in vivo. There further is a need for the development of spin trapping compounds which are capable of forming spin adducts with radicals which are readily identifiable spectrophotometrically.
It is therefore an object of the invention to provide methods for synthesizing spin trapping compounds capable of reacting with free radicals in biological systems. It is another object of the invention to provide spin trapping compounds which are capable of forming stable spin adducts with characteristic and readily identifiable electron paramagnetic resonance ("EPR") spectra. It is yet another object of the invention to provide spin trapping compounds which can be used in diagnostic and therapeutic applications.